Abstract
Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 A resolution. BVP adopts the characteristic cysteine-phosphatase alpha/beta fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif (118)HCTHGXNRT(126) in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.
Highlights
MRNA 5Ј cap formation is initiated by hydrolysis of the ␥-phosphate of 5Ј-triphosphate-terminated pre-mRNA
Structural, biochemical, and mutational results showed that despite sharing a HCXXXXXR(S/T) P-loop motif, a phosphoenzyme intermediate, and a core ␣/ fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by Mce1 and its baculovirus homologue Baculovirus RNA 5-triphosphatase (BVP) 1 differs from that used by phosphoprotein phosphatases to hydrolyze phosphomonoesters
The structure of BVP was determined by molecular replacement using the structure of the RNA triphosphatase domain of Crystal Structure of Baculovirus Phosphatase
Summary
MRNA 5Ј cap formation is initiated by hydrolysis of the ␥-phosphate of 5Ј-triphosphate-terminated pre-mRNA. We reported previously [3] the crystal structure of the RNA triphosphatase domain of mouse capping enzyme Mce at 1.65 Å resolution. Structural, biochemical, and mutational results showed that despite sharing a HCXXXXXR(S/T) P-loop motif, a phosphoenzyme intermediate, and a core ␣/ fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by Mce and its baculovirus homologue BVP 1 differs from that used by phosphoprotein phosphatases to hydrolyze phosphomonoesters. BVP displays primary structure similarity to the N-terminal RNA triphosphatase domain of Mce. Unlike the cellular capping enzyme, which hydrolyzes only the ␥-phosphate of a triphosphate-terminated substrate, BVP can hydrolyze the -phosphate of either diphosphate-terminated RNA or free NDPs [6, 7]. PIR1like proteins are present in many metazoan proteomes, but their functions are not known [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
